Members of the genus Flaviviruses, such as West Nile, Japanese encephalitis, tick-borne encephalitis, yellow fever, and dengue virus, are human pathogens that cause significant morbidity and mortality in ever-widening geographic areas of the world. Currently, there are few vaccines and no available efficacious antiviral therapies for flaviviruses. A Mendelian dominant allele of the Flv gene that was first discovered in mice in the 1920's reduces the replication efficiency of all members of the genus Flavivirus so far tested and confers resistance to flavivirus-induced disease. Resistant mice display no symptoms even after an intracerebral injection of a flavivirus dose that kills 100% of congenic susceptible mice. Viral attachment and entry are not blocked but intracellular viral RNA levels and extracellular virus production are reduced in resistant cells. The Flv locus was subsequently identified as the Oas1b gene. Susceptible mice express a truncated Oas1b protein due to the presence of a premature stop codon. Replacement of the susceptible Oas1b allele by knock-in of the Oas1b resistance allele using targeted homologous recombination converted susceptible C57BL/6 mice to the resistant phenotype. Oas1b is a member of the 2-5A synthetase family but the products of both alleles of this gene are inactive synthetases. The unique flavivirus-specific antiviral activity of Oas1b is not dependent on the canonical RNase L pathway. The recent identification of cell partners for Oas1b has provided new directions for gaining an understanding of this novel, natural, flavivirus-specific antiviral mechanism. Preliminary data for one of the Oas1b binding partners, ABCF3, indicate that it is specifically involved in the flavivirus-resistance mechanism and that it may also be involved in regulating the canonical Oas-RNase L pathway. Functional contributions of the Oas1b binding partners to the flavivirus-specific resistance phenotype and for one of the partners, a contribution to a broad spectrum antiviral pathway will be studied under three specific aims. Aim 1: Characterization of the resistance phenotype in various types of cell cultures and functional analyses of the involvement of Oas1b partner proteins in the flavivirus resistance mechanism. Aim 2: Characterization of the resistance phenotype in mouse brains and analysis of the requirement of ABCF3 for the Oas1b-mediated, flavivirus-specific resistance phenotype in mice. Aim 3: Functional analysis of the interaction of ABCF3 with the active 2-5A synthetase Oas1a. This project will provide new knowledge about a novel, naturally-occurring, genetically-controlled, flavivirus-specific disease resistance mechanism in mice as well as reveal new information about the unique step in the flavivirus replication cycle that is affected by this resistance mechanism. This information is expected to identify new cell targets for the development of pan-flavivirus antiviral therapeutics. New insights about the regulation of the Oas-RNase L pathway will also be obtained.